Molecules, compositions, methods and kits for applications associated with flaviviruses

ABSTRACT

A method for controlling a flavivirus entry into a cell, kits for assaying the flavivirus entry into the cell, and methods of treating and preventing flaviviruses infections are disclosed, together with vaccine and pharmaceutical compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/197,966, filed Aug. 25, 2008, which is a divisional of U.S. patentapplication Ser. No. 10/769,565, filed Jan. 29, 2004, issued as U.S.Pat. No. 7,449,321 on Nov. 11, 2008; which application is acontinuation-in-part of U.S. patent application Ser. No. 10/763,450filed on Jan. 22, 2004, now abandoned; which application claims thebenefit under 35 U.S.C. §119(e) of U.S. Provisional Patent ApplicationNo. 60/442,157, filed Jan. 22, 2003, which applications are incorporatedherein by reference in their entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 920094_(—)401D2_SEQUENCE_LISTING.txt. The textfile is 64 KB; it was created on Jan. 26, 2011; and it is beingsubmitted electronically via EFS-Web, concurrent with the filing of thespecification.

BACKGROUND

1. Technical Field

The present disclosure relates to the field of virology, and inparticular to molecules, compositions, methods and kits for applicationsassociated with flaviviruses.

2. Description of the Related Art

The family Flaviviridae contains at least 70 arthropod-transmittedviruses, many of which infect humans and other vertebrates. A subgroupof the Flaviviridae family, the Japanese encephalitis serocomplex,includes West Nile Virus, St. Louis encephalitis, Murray Valleyencephalitis and kunjin viruses. West Nile virus, in particular, is mostcommonly found in Africa and the Middle East.

All flaviviruses, including West Nile Virus, St Louis encephalitis,dengue, Japanese encephalitis, yellow fever and kunjin viruses sharesimilar size, symmetry and appearance. Despite the fact thatflaviviruses may use different process to enter a host cell, such asendocytotis (described for West Nile Virus and Kunjin Virus) and directfusion of the cell (described for dengue and Encephalitis Virus), entryof all flaviviruses into the host-cell involves an interaction betweenthe virus and a receptor of the cell.

Several studies have shown that the viral envelope protein offlaviviruses plays a crucial role in mediating virus-host cellularreceptor interaction. Based on crystallography data of tick-borneencephalitis flavivirus viral envelope protein, Rey and colleagues(1995) noted that each viral envelope protein monomer is folded into 3distinct domains. A central domain I is the antigenic domain thatcarries the N-glycosylation site. Domain II of the viral envelopeprotein is believed to be responsible for pH-dependent fusion of theviral envelope protein to the endosomal membrane during uncoating, anddomain III is important for flavivirus binding to host cells.

With reference to West Nile virus, Beasley and Barrett (2002) focused onthe importance of subportions of the Domain III in West Nile virology.They identified and mapped as epitopes, portions of Domain III whoseneutralization by single monoclonal antibodies may result inneutralization of the virus.

The specific interaction between flaviviruses and a vertebrate cellsurface or surface membrane receptor is unknown. Without knowledge ofthe details of this interaction, it has proven difficult to specificallytreat or prevent the disease. Therefore, there is clearly a need for theidentification of the cell receptor, as well as the domain/s of theflavivirus that mediate their respective interactions.

BRIEF SUMMARY

The present disclosure overcomes the problems and disadvantages of theprior art.

According to a first aspect of the present disclosure, a method forcontrolling a flavivirus entry into a cell is disclosed, comprisingadministering to the cell an agent functionally interfering with aflavivirus receptor protein, the receptor protein being an integrin.

The integrin preferably comprises integrin subunit β3 or integrinsubunit αV, and most preferably is an αVβ3 integrin. The agentfunctionally interfering with a flavivirus receptor protein ispreferably a functional blocking antibody against the integrin, or acompetitive ligand for the integrin, in particular an RGD peptide or anatural ligand selected from the group consisting of fibronectin,vitronectin, laminin and chrondriotin.

According to a second aspect, a method for controlling flavivirus entryinto a cell is disclosed, comprising administering to the cell an agentinterfering with the expression of a flavivirus receptor protein, thereceptor protein being integrin.

The agent interfering with the expression of the flavivirus receptorprotein is preferably a siRNA against the integrin.

According to a third aspect, a kit for controlling flavivirus entry intoa cell is disclosed, comprising: the flavivirus; an agent functionallyinterfering with an integrin. The flavivirus and the agent are to beused in the method disclosed herein.

Preferably, the agent functionally interfering with an integrin is afunctional blocking antibody against the integrin or a competitiveligand for the integrin.

According to a fourth aspect, a further kit for controlling flavivirusentry into a cell, is disclosed, comprising: the flavivirus; and anagent interfering with expression of an integrin. The flavivirus and theagent interfering with the expression of the integrin are to be usedaccording to the method disclosed herein.

An agent functionally interfering with an integrin may also be includedin the kit disclosed herein and is to be used according to the methodcomprising its administration disclosed herein. The agent interferingwith the expression of an integrin is preferably an SiRNA against theintegrin.

According to a fifth aspect, a further method for controlling aflavivirus entry into a cell is disclosed, comprising administering tothe cell an agent functionally interfering with an ATPase in the plasmamembrane of the cell, preferably a functionally blocking antibodyagainst the ATPase.

According to sixth aspect, a kit for controlling a flavivirus entry intoa cell is disclosed, comprising: the flavivirus; and an agentfunctionally interfering with an ATPase located in the plasma membraneof the cell. The flavivirus and the agent are to be used according tothe method disclosed herein.

An agent functionally interfering with an integrin and/or an agentinterfering with the expression of an integrin may also be included inthe kit and are to be used according to the methods comprising therespective administration herein also disclosed.

According to a seventh aspect, a method for controlling a flavivirusentry into a cell is also disclosed, which comprises administering tothe cell an agent functionally interfering with a flavivirus receptorprotein, the receptor protein being a neurotensin receptor.

Preferably, the agent functionally interfering with a flavivirusreceptor protein is a functional blocking antibody against theneurotensin receptor, or a competitive ligand for the neurotensinreceptor, in particular neurotensin.

According to an eighth aspect, a kit for controlling a flavivirus entryinto a cell is disclosed, comprising: the flavivirus; and an agentfunctionally interfering with a neurotensin receptor in the cell. Theflavivirus and the agent are to be used according to the methoddisclosed herein.

An agent functionally interfering with an integrin, an agent interferingwith the expression of an integrin and/or an agent functionallyinterfering with an ATPase in the plasma membrane of the cell may alsobe included in the kit and are to be used according to the methodscomprising the respective administration herein also disclosed.

According to a ninth aspect, a method for controlling a flavivirus entryinto a cell is disclosed, the flavivirus exhibiting a flavivirusenvelope protein, the flavivirus envelope protein comprising a domainIII, the method comprising administering to the cell an agentfunctionally interfering with the domain III of the flavivirus envelopeprotein. Preferably the domain III of the virus comprise a portionhaving a sequence substantially homologous to SEQ ID NO: 19 or SEQ IDNO: 21.

According to a further aspect, a method for treating a flavivirusinfection in a vertebrate is disclosed, the flavivirus exhibiting aflavivirus envelope protein, the flavivirus envelope protein comprisinga domain III. The method comprises administering to the vertebrate apharmaceutically effective amount of an agent functionally interferingwith the domain III of the envelope protein of the flavivirus, able toinhibit the entry of the flavivirus in the cell.

According to a further aspect, a pharmaceutical composition for thetreatment of a flavivirus infection in a vertebrate is disclosed, theflavivirus exhibiting an envelope protein comprising a domain III. Thepharmaceutical composition comprises a pharmaceutically effective amountof an agent interfering with the domain III of the envelope protein ableto inhibit the entry of the Flavivirus in the host cell and apharmaceutically acceptable carrier, vehicle or auxiliary agent.

Both in the method of treating and pharmaceutical composition, the agentis preferably one of the functionally interfering agent able to inhibitthe entry in the cell mentioned above. In particular, a functionalblocking antibody against the domain III, preferably a polyclonalantibody, an integrin protein, preferably comprising one or both of thesubunits αV and β3, or a neurotensin receptor protein or an ATPase,preferably an F-ATPase or V-ATPase, or portions thereof may be used.

According to a further aspect, a method for inducing immunity to aflavivirus in a vertebrate susceptible to the infection of theflavivirus is disclosed, the flavivirus exhibiting an envelope proteincomprising a domain III. The method comprises administering to thevertebrate an immunogenic amount of a polypeptide comprising the domainIII, of the envelope protein of the flavivirus, preferably comprising aportion substantially homologous to SEQ ID NO: 19 or SEQ ID NO: 21.

According to a further aspect, a vaccine for a flavivirus, theflavivirus exhibiting an envelope protein comprising a domain III, isdisclosed. The vaccine comprises as an active agent a polypeptidecomprising the domain III of the envelope protein of the flavivirus.

According to a further aspect, a method for diagnosing a flavivirusinfection in a vertebrate susceptible to infection by the flavivirus isdisclosed, comprising contacting a sample tissue from the vertebrate,with an integrin or neurotensin protein associated with an identifier;and detecting presence or absence of a flavivirus-integrin complex orflavivirus-neurotensin complex by detecting presence of the identifier.

According to a further aspect, a kit for the diagnosis of flavivirusinfection in a vertebrate, susceptible to be infected with theflavivirus, the flavivirus exhibiting an envelope protein comprisingdomain III is disclosed. The kit comprises at least one agent able tobind the domain III, associated with an identifier, and one or morereagents able to detect the identifier. The agent able to bind domainIII and the reagents are to be used according to the diagnostic methoddisclosed above.

According to a further aspect, a diagnostic method to analyze a cellsusceptibility to flavivirus infection, is disclosed, comprisingcontacting the cell with an identifier for the presence or expression ofan integrin, neurotensin receptor and/or ATP-ase and detecting thepresence of the identifier associated to presence or expression of anintegrin, neurotensin receptor and/or ATP-ase in the cell.

According to another aspect, a kit to analyze cell susceptibility toflavivirus infection is disclosed comprising an identifier for thepresence or expression of an integrin, neurotensin receptor and orATP-ase, and a reagent able to detect the presence of the identifier;the identifier and the reagent to be used in the method disclosed above.

According to a further aspect, an isolated and purified plasma membranepolypeptide of approximately 105 KDa comprising a sequence substantiallyhomologous to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, andSEQ ID NO:5 is disclosed.

In the methods, kits, composition and vaccine disclosed herein, theflavivirus is preferably a member of the Japanese encephalitisserocomplex, in particular West Nile Virus, and the vertebrate ispreferably a mammal, in particular a human being.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

In the present disclosure, reference will be made to the enclosedFigures, which provide non-limiting examples of the inventivecompositions, method and kits devised by the inventors.

FIG. 1A shows a diagram reporting the effect of phospholipases andproteases treatment on West Nile virus binding molecules present on thesurface of intact Vero cells. The y-axis shows the inhibition of WestNile virus entry expressed as the number of log unit inhibition withrespect to untreated samples. On the x-axis, the dosage of the substanceadministered is reported. PLA=Phospholipase A₂, PLC=Phospholipase C,PLD=Phospholipase D

FIG. 1B shows a diagram reporting the effect of enzyme, sodium periodateand lectin treatment on West Nile virus binding molecules present on thesurface of intact Vero cells. The y-axis shows the inhibition of WestNile virus entry expressed as the number of log unit inhibition withrespect to untreated samples. On the x-axis, the dosage of substanceadministered is reported. EH=Endoglycosidase H, OG=O-Glycosidase,MA=α-Mannosidase; FU=α-Fucosidase; HepI=Heparinase I; HepIII=HeparinaseIII; SP=Sodium Periodate; WGA=Wheat Germ Agglutinin;PHA=phytohemagglutinin; ConA=Concanavalin-A.

FIG. 2A shows the results of a Virus Overlay Protein Binding Assay(VOPBA) performed on plasma membrane proteins extracted from Vero cellsbefore purification under non-denaturing conditions (Lane 1) as comparedto those of proteins purified from the supernatants of uninfected cells(Lane 2). Molecular size markers are indicated on the left side of theFigure by arrows.

FIG. 2B shows the results of a VOPBA performed on plasma membraneproteins extracted from N2A cells extracted before purification undernon-denaturing conditions (Lane 1) as compared to those of proteinspurified from the supernatants of uninfected cells (Lane 2). Molecularsize markers are indicated on the left side of the Figure by arrows.

FIG. 3 shows results of a VOPBA performed on plasma membrane proteinsfrom Vero cells and N2A cells after papain treatment (Lane 2—Vero cells;lane 4—N2A), as compared to those of the untreated cells (Lane 1—Verocells; lane 3—N2A). Molecular size markers are indicated on the leftside of the Figure by arrows.

FIG. 4 shows results of a VOPBA performed on plasma membrane proteinsfrom Vero cells after papain treatment (Lane 2—after 0 hours; lane3—after 2 hours; Lane 4—after 4 hours), as compared to those ofuntreated cells (Lane 1) and those of treated cells further subjected tocycloheximide treatment (Lane 2—after 0 hour; lane 3—after 2 hours; Lane4—after 4 hours). Molecular size markers are indicated on the left sideof the Figure by arrows.

FIG. 5A shows results of a VOPBA performed on plasma membrane proteinsextracted from Vero cells (Lanes 1 to 4) and N2A cells (Lanes 5 to 8)either left untreated (Lanes 1 and 5) or subjected to α-mannosidase(Lanes 2 and 6), Endoglycosidase H (Lanes 3 and 7) and O-glycosidase(Lanes 4 and 8). A molecular size marker is indicated on the right sideof the Figure by an arrow.

FIG. 5B shows results of a VOPBA performed on plasma membrane proteinsobtained from Vero cells pretreated with lectins, concananvalin-A [Lane1 (untreated), Lane 2 (10 μg/ml) and Lane 3 (100 μg/ml)] andphytohemagglutinin [Lanes 4 (untreated), Lane 5 (10 μg/ml) and Lane 6(100 μg/ml)]. A molecular size marker is indicated on the right side ofthe Figure by an arrow.

FIG. 5C shows results of a VOPBA performed on plasma membrane proteinsobtained from Vero cells either left untreated (Lane 1) or treated with0.1 mM (Lane 2), 1 mM (Lane 3) and 10 mM (Lane 4) of sodium periodate.Molecular size markers are indicated on the left side of the Figure byarrows.

FIG. 6 shows results of a VOPBA performed on membrane proteins from Verocells and N2A cells after β-mercaptoethanol treatment (Lane 2—Verocells; lane 4—N2A) as compared to those of the untreated cells (Lane1—Vero cells and Lane 3—N2A cells). Molecular size markers are indicatedon the left side of the Figure by arrows.

FIG. 7 shows results of a Western Blotting performed on membraneproteins from plasma membrane extracts of Vero cells. Incubation ofseparated membrane proteins with the preimmune serum (Lane 1) and theanti-105-kDa protein polyclonal antibodies at a dilution of 1:500 (Lane2) was performed. Molecular size markers are indicated on the left sideof the Figure by arrows.

FIG. 8A shows Vero cells processed for immunofluorescence and confocalmicroscopy. The arrows indicate the 105-kDa proteins distributed alongthe plasma membrane as shown by the red staining.

FIG. 8B shows apical localization of the 105-kDa membrane proteins onZ-section (cross-section) of polarized Vero C1008 epithelial cells. Thearrows indicate the 105-kDa membrane proteins at the apical surface ofthe cells.

FIG. 9 shows Immunogold-labeling of the 105-kDa proteins oncryo-sections of Vero cells. The arrow indicates the binding of WestNile virus to the 105-kDa membrane protein indicated by arrows at thesite of virus attachment.

FIG. 10 shows a VOPBA of Vero plasma membrane protein pre-incubated withanti-105 kDa murine antibodies (Lane 1) compared to that of untreatedcells (Lane 2). Molecular size markers are indicated on the left side ofthe Figure by arrows.

FIG. 11A is a diagram showing the inhibition of West Nile Virus bindingin Vero cells pre-incubated with functional blocking integrinantibodies. On the x-axis, the concentration of functional blockingintegrin antibodies is shown. On the y-axis, the percentage reduction invirus infection is shown. INTB1=Integrin β1, INTB2=Integrin β2,INTB31=integrin beta 3, INTB32=integrin beta 3 subunit, INTB4=integrinβ4, INTB5=Integrin β5, ALPHAV=Integrin αV, ALPHA V/B3=Integrin αVβ3,ALPHA5/B5=Integrin α5 β5.

FIG. 11 B is a diagram showing the inhibition of West Nile Virus entryin Vero cells pre-incubated with functional blocking integrinantibodies. On the x-axis the concentration of functional blockingintegrin antibodies is shown. On the y-axis the percentage reduction invirus infection is shown. INTB1=monoclonal antibodies against Integrinβ1, INTB2=Integrin β2, INTB31=monoclonal antibodies against integrinbeta 3 subunit purchased from Chemicon, USA, INTB32=monoclonalantibodies against integrin beta 3 subunit purchased from Santa CruzBiotech, USA., INTB4=monoclonal antibodies against integrin β4,INTB5=monoclonal antibodies against Integrin β5, ALPHAV=monoclonalantibodies against Integrin αV, ALPHA V/B3=monoclonal antibodies againstIntegrin αVβ3, ALPHA5/B5=monoclonal antibodies against Integrin α5 β5.

FIG. 12 is a diagram showing the inhibition of Japanese EncephalitisVirus entry into Vero cells by functional blocking integrin antibodies.On the x-axis the integrin blocked by the specific anti integrinantibody is shown. On the y-axis the % inhibition in virus entry isshown. INTB1=monoclonal antibodies against Integrin β1; INTB2=monoclonalantibodies against Integrin β2, INTB31=monoclonal antibodies againstintegrin beta 3 subunit purchased from Chemicon, USA, INTB32=monoclonalantibodies against integrin beta 3 subunit purchased from Santa CruzBiotech, USA.; INTB4=monoclonal antibodies against integrin β4;INTB5=monoclonal antibodies against Integrin β5, ALPHAV=monoclonalantibodies against Integrin αV; ALPHA V/B3=monoclonal antibodies againstIntegrin αVβ3; ALPHA5/B5=monoclonal antibodies against Integrin α5 β5.

FIG. 13 is a diagram showing the effect of divalent cations chelatorEDTA on the entry of West Nile Virus (WNV) and Japanese EncephalitisVirus (JEV) in Vero cells. On the x-axis, the concentration of EDTAadministered is shown. The y-axis shows percentage reduction of virusinfection.

FIG. 14 is a diagram showing a competitive entry study of West NileVirus (WNV) and Japanese Encephalitis Virus (JEV) with physiologicalligands on Vero cells pretreated with such ligands. On the x-axis, thecompetitive ligands used, are shown. On the y-axis, the percentagereduction of virus infection is shown. RGE1=RGE-peptide;RGD1=RGD-peptide; FIBRO1=Fibronectin; VITRO1=Vitronectin; LAMININ1=laminin; CHONSUL1=Chrondriotin; Heparin=Heparin.

FIG. 15 shows distribution and localization of integrin αVβ3 (A) and 105KDa plasma membrane glycoprotein (B) in Vero cells by immunofluroscencestaining.

FIG. 16 shows results of gene silencing of integrin αVβ3 subunits αV (B)and β3 (D) in Vero cells compared to control for αV (A) and β3 (C).Presence of the subunits is shown by immunofluorescence staining.

FIG. 17 is a diagram showing the effects of down-regulation of integrinαV and β3 subunits to the entry of WNV into Vero cells. On x-axisconcentration of siRNA used is reported. On y-axis percentage inhibitionof WNV entry is reported. INTAlpha V1=Integrin alpha V subunit region 1,INTalpha2=Integrin alpha V subunit region 2, INTB31=Integrin beta 3subunit region 1, INTB32 =Integrin beta 3 subunit region 2,GAPDH=Glyceraldehyde-3-phosphate dehydrogenase.

FIG. 18 is a diagram showing effects of administration of antibodiesagainst ATPases. On the x-axis, the antibodies against the respectiveprotein are indicated. On the y-axis, the percentage reduction of virusentry is indicated. ATPB1=monoclonal antibodies against plasma membraneATPase beta subunit, ATPB2=polyclonal antibodies against plasma membraneATPase beta subunit, ATPA1=monoclonal antibodies against plasma membraneATPase alpha subunit, ATPA2=polyclonal antibodies against plasmamembrane ATPase alpha subunit, CALTYPE1=monoclonal antibodies againstcalcium dihydropyridine receptor alpha, CALTYPE2=monoclonal antibodiesagainst calcium dihydropyridine receptor beta, VATPASE1=monoclonalantibodies against VATPase E, VATPASE2=monoclonal antibodies againstVATPase.

FIG. 19 is a diagram showing blockage of WNV entry by antibodies againstneurotensin receptor, in Vero cells (black column) and in A172neuroblastoma cells (grey column). On the x-axis, the concentration ofanti-neurotensin receptor is shown. The y-axis shows the percentagereduction of virus entry.

FIG. 20 is a diagram showing WNV competiting binding of neurotensinreceptor with its natural ligand in A172 neuroblastoma cells. On thex-axis, the concentration of neurotensin administered before incubationof the cells with WNV is shown. The y-axis shows percentage inhibitionof virus entry.

FIG. 21 shows immunofluorescence assays performed in A172 cells (A), andin A172 cells transfected with pSilencer-siRNA neurotensin receptor (B).

FIG. 22 shows results of a Western Blotting carried out with monoclonalantibodies against (A) E-protein of WNV and (B) anti-His lysate, wholecell lysate where domain II of WNV was cloned and expressed asHis-tagged fusion protein. (A) lane 1=IBV nucleocapsid protein; lane2=Dengue infected whole cells lysate; lane 3=buffer; lane 4=DIIIprotein; and lane 5=West Nile virus infected whole cell lysate. (B) lane1=IBV nucleocapsid protein; lane 2=buffer; lane 3=DIII protein; and lane4=West Nile virus infected whole cell lysate. Molecular size markers areindicated on the left side of the Figure by arrows.

FIG. 23 is a diagram showing results of competitive inhibition of WNVand Dengue virus entry with soluble recombinant WNV envelope DIII. Onthe x-axis, concentration of inhibitor(s) is reported. On the y-axis,percentage inhibition of virus entry is reported.

FIG. 24 shows production of murine polyclonal antibodies againstrecombinant DIII protein. Lane 1=recombinant DIII protein with anti-DIIIprotein polyclonal murine antibodies; and lane 2=DIII protein withpre-immunized murine sera. Molecular size markers are indicated on theleft side of the Figure by arrows.

FIG. 25 is a diagram showing results of plaque neutralization of WNVwith murine polyclonal antibodies against envelope DIII protein. On thex-axis, various grades of dilution used are reported. On the y-axis,percentage inhibition of virus infection is reported.

DETAILED DESCRIPTION OF THE DISCLOSURE

A method for controlling the entry of a flavivirus into a cell isdescribed. In particular, the method is based on the identification ofintegrins as receptors which mediate entry of the flavivirus into thecell.

In their quality as flavivirus receptors, integrins have been found tosurprisingly mediate entry of a wide number of flaviviruses usingdiverse processes to enter the host cell, such as endocytosis and cellfusion. In particular, integrins have been shown to mediate the entry inthe cell of flaviviruses belonging to the Japanese EncephalitisSerocomplex, in particular West Nile Virus, St. Louis encephalitis,Murray Valley encephalitis, as well as the entry of other flavivirusessuch as dengue and kunjin.

Additionally, the activity of integrins as flavivirus receptorsdisclosed herein applies to a wide range of cell systems, includingbrain cells, and to a wide number of organisms, including vertebratesand human beings.

Integrins have been identified to be flavivirus receptors through aseries of experiments extensively described in the examples that follow.

In particular, in a first series of experiments, described in theexamples 1 to 9, the receptor has been first identified as a proteasesensitive glycoprotein with complex N-linked sugars containing α-mannoseresidues, localized on the cell membrane. In particular, a 105-KDaglycoprotein exhibiting all these properties has been isolated (seeexample 2).

The experiments have been carried out on Vero cells and N2A cells. Bothare cells lines highly permissive for West Nile Virus, which has beenused as a model for flaviviruses. Vero cells is a Green Monkey cell lineand has been used as a cell system to isolate the receptor for WNV. N2Ais a mouse derived brain cell line, which has been used as analternative to human brain cells, since West Nile virus has showntropism to brain cells during infection in mammals.

West Nile virus, in particular the Sarafend strain, has been used inthese experiments. West Nile Virus has been used as a representative ofthe flavivirus and in particular of the flavivirus belonging to theJapanese Encephalitis Serocomplex group. Other flaviviruses of theJapanese Encephalitis Serocomplex group, including kunjin, as well asflaviviruses not belonging to such group, such as dengue, were also usedto extend the analysis on the integrins' activity as flavivirusreceptors to the entire flavivirus family.

The experiments extensively described in examples 10 and 11 confirm the105 KDa glycoprotein's ability to act as a receptor for West Nile andother flaviviruses, in particular those of the Japanese Encephalitisserocomplex group, including St. Louis encephalitis, Murray ValleyEncephalitis, as well as dengue and kunjin. In particular in view ofthose results, a significant receptor activity for any flavivirusesbelonging to the flavivirus family is expected. In particular, receptoractivity for the flavivirus having an E protein substantially homologousto the E protein of a member of the Japanese Serocomplex group, such asyellow fever and tick borne, is expected.

Further analysis of the 105 KDa protein has confirmed location of thereceptor on the membrane (Example 12) and the fact that the receptor isan integrin (Example 13). In particular, further experiments assayingflavivirus entry inhibition, sequencing of the 105 KDa protein and VirusOverlay Protein Binding Assays, described in examples 11 to 17, haveidentified the 105 KDa protein as a αVβ3 integrin and a significantreceptor activity of integrins comprising subunits other than αV and β3.

In particular, experiments reported in examples 13 to 17 showed asignificant ability of integrins to act as receptors for flaviviruseswherein subunits have an independent ability to act as a receptor forthe virus. In particular, a particularly significant ability of integrinsubunits αV and β3 to act as a receptor was observed. However a betterefficiency is obtained when both subunits are present.

These results have been obtained in Vero cells and N2A cells. However,since integrins are expressed in most types of cells, including braincells of vertebrates, and since flaviviruses, and in particular WNV,have been shown to afflict a range of other mammals such as horses andhumans, as well as other vertebrates such as birds, the scope of theseresults can be extended to these other systems as well.

The administration of an agent able to functionally interfere with theintegrin has been shown to affect the flavivirus entry in the host cell(see Examples 1-22).

Therefore, the present disclosure shows that administration of an agentthat functionally interferes with integrin affects flavivirus entry inthe host cell. In particular, agents able to interfere with thefunctionality of the attachment domain of the integrin are functionallyinterfering agents of this disclosure.

Functionally interfering agents can enhance or inhibit the integrinfunctionality. In particular, interfering agents able to functionallyinhibit integrins, such as functional blocking antibodies andcompetitive ligands, are considered to be a functionally interferingagent able to inhibit the entry of flavivirus.

Preferably, the functionally blocking antibodies are polyclonalantibodies, in particular against the 105 KDa protein, the integrinsubunits αV, β3, αVβ3 or αVβ35.

The competitive ligand can be a natural ligand, such as for examplefibronecitin, vitronectin or laminin, or a synthetic ligand, for exampleRGD peptide or chemically synthesized peptides that are complementary tothe binding region in the integrin, which ligand can be identified andmanufactured by a person skilled in the art, based on the informationprovided in the present application.

Proteases such as papain, glycosidases, lectins, and cycloheximide arealso considered functionally interfering agents able to inhibit theintegrin functionality.

These functionally interfering agents were used at a range ofconcentrations that are non-cytotoxic to the cells used in our system.In particular, functionally inhibitors of the integrin can beadministered at following amounts: papain 10-50 mUnit/ml; Lectin and inparticular Concanavalin-A phytohemagglutinin) 100-1000 μg/ml;cycloheximide about 100 μg/ml; Endoglycosidase 10-100 mUNIT/ml;O-glycosidase 0.1-1 mUNIT/ml; mannosidase 100-1000 μg/ml; Fucosidase10-100 mUNIT/ml.

With reference to competitive ligands, effective concentrations thatwill block the entry of West Nile virus will be in the ranges thatfollows: RGE peptide: 0.01-30 μg/ml; RGD peptide: 0.01-30 μg/ml;Fibronectin: 0.01-40 μg/ml; Vitronectin: 0.01-40 μg/ml; Laminin: 0.01-40μg/m; Chrondriotin sulphate: 0.01-40 μg/ml; Heparin: 0.01-40 μg/ml.These ligands can be administered before an infection occurs or duringan infection to block the entry of the subsequent newly produced virusprogeny from entry. The ligand needs to be incubated with the cells forat least 30 min for effective binding to the cells and can be presentfor more than 1 hr. The temperature for incubating the ligand with cellscan be in the range of about 4° C. to 40° C.

With reference to the antibodies gainst integrin subunits, the effectiveconcentrations of the functional blocking integrin (all the integrinused) antibodies that will block the entry of West Nile virus will be inthe range of about 0.025 μg/ml-40 μg/ml. These antibodies can beadministered before an infection occurs or during an infection to blockthe entry of the subsequent newly produced virus progeny from entry. Theantibodies are preferably incubated with the cells for at least 10 minfor effective binding to the cells and can be present for more than 1hr. The temperature for incubating the antibodies with cells can be inthe range of about 4° C. to 40° C.

The present disclosure also shows that agents interfering with theexpression of the integrin are able to affect the entry of theflavivirus into the host cell.

In particular, interference with the expression of the integrin mayresult in inhibition or enhancement of such expression. For example, asilencer or preferably a short interfering RNA is suitable for use withthe present invention and allows flavivirus activity to be inhibited.Other agents interfering with the expression of the integrin areidentifiable by a person skilled in the art based on the informationprovided in the present application.

The present disclosure shows that entry of flavivirus is also affectedby administration of an agent functionally interfering with thefunctionality of an ATPase in the cell. In particular the ATPases asintended herein include but are not limited to the plasma membraneassociated ATPases (known as F-ATPases) and the vacuolar ATPases thatcan be localized to the plasma membrane as well as the membrane ofendocytic vesicles and lysosomes.

Functionally interfering agents can enhance or inhibit the ATP-asefunctionality. In particular, interfering agents able to functionallyinhibit the ATPases, such as functional blocking antibodies andcompetitive ligands, are examples of functionally interfering agentsable to inhibit the entry of flavivirus.

Preferably, the functionally blocking antibodies are monoclonal andpolyclonal antibodies in particular against extracellular subunits ofATPases (plasma membrane) as well as V-ATPases. It has been shown thatsuch antibodies are particularly effective in inhibiting flavivirusentry (See Example 19).

The competitive ligand can be a natural ligand or a synthetic ligand,such as chemically synthesized peptides that are complementary tobinding regions in the ATPase, which can be identified and manufacturedby a person skilled in the art based on the information provided in thepresent application.

Administration of an agent that functionally interferes with aneurotensin receptor affects flavivirus entry in the host cell isdisclosed. Neurotensin receptors are present in the brain andgastrointestinal tract and are involved in neurotransmission.

Functionally interfering agents can enhance or inhibit the neurotensinfunctionality. In particular, interfering agents able to functionallyinhibit a neurotensin receptor, such as functional blocking antibodiesand competitive ligands, are examples of functionally interfering agentsable to inhibit the entry of flavivirus

Preferably, the functionally blocking antibodies are monoclonal andpolyclonal antibodies, directed, in particular, against theextracellular portion of the neurotensin receptor.

More specifically, the inventors show that, in particular competitivenatural ligands such as neurotensin and antibodies against the receptor,inhibit the entry of the virus (See Examples 20-21). Other competitiveligands known to compete with neurotensin for neurotensin receptor canused. For example, Neuromedin N, 8 bromo-cAMP, IBMX and forskolin arecompetitive ligands competing with neurotensin for the neurotensinreceptor (Shi & Bunney 1992), which are expected to inhibit the entry ofthe virus.

The competitive ligand can also be a synthetic ligand, such aschemically synthesized peptides that are complementary to the bindingregion in the neurotensin receptor. Also in this case, ligands can beidentified and manufactured by a person skilled in the art based on theinformation provided in the present application.

The present disclosure provides a further method for interefering withflavivirus entry. According to this further method, domain III in theenvelope protein of the flavivirus has been found to bind the flavivirusreceptor in the host cell.

Experiments of competitive binding between the domain III of the virusenvelope protein, expressed in soluble form and West Nile and dengueviruses, extensively reported in Example 22, show that the domain III ofthe envelope protein is the attachment domain of the envelope proteinfor the receptor protein in the cell.

Therefore, administration of an agent that functionally interferes withdomain III of the envelope protein also affects flavivirus entry in thehost cell. The agent functionally interfering with the domain IIIactivity can enhance or inhibit the functionality of domain III.Preferably, the functionally interfering agent is able to inhibit thefunctionality of domain III. In particular, agents such as a competitiveligand of domain III or an antibody against domain III, are functionallyinterfering agent able to inhibit the functionality of the domain III.

In particular, the competitive ligand can be a competitive naturalligand of domain III such as an integrin, and more specifically anintegrin comprising at least one of αV and β3 integrin subunits,preferably an integrin comprising both subunits αV and β3. Thecompetitive natural ligand can be also a neurotensin receptor or anATPase, preferably an F-ATPase or V-ATPase or a molecule substantiallyhomologous thereto. The competitive ligand can also be a syntheticligand, such as chemically synthesized peptides that are complementaryto domain III or the binding region in the integrin or neurotensin orATPase, which can be identified and manufactured by a person skilled inthe art based on the information provided in the present application.The antibody against the domain III is preferably a polyclonal antibodyagainst domain III, most preferably a functional blocking polyclonalantibody against domain III.

An antibody against a membrane 105 KDa polypeptide having a sequencesubstantially homologous to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO: 4 and SEQ ID NO:5 is also here disclosed.

A kit of parts for controlling and assaying entry of the flavivirus in acell is also disclosed, comprising the flavivirus and at least one amongthe functionally interfering agents here described and/or agentsinterfering with the integrin expression.

The flavivirus and the at least one among the functionally interferingagents are comprised in the kit independently in one or morecompositions wherein each is in a composition together with a suitablevehicle carrier or auxiliary agent.

In particular, the flavivirus and functionally interfering agents orexpression-interfering agents interfering with the expression can beprovided in the kits, with suitable instructions and other necessaryreagents, in order to perform the methods here disclosed. The kit willnormally contain the compositions in separate containers. Instructions,for example written or audio instructions, on paper or electronicsupport such as tapes or CD-ROMs, for carrying out the assay, willusually be included in the kit. The kit can also contain, depending onthe particular method used, other packaged reagents and materials (i.e.wash buffers and the like).

Further details concerning the identification of the suitable carrieragent or auxiliary agent of the compositions, and generallymanufacturing and packaging of the kit, can be identified by the personskilled in the art upon reading of the present disclosure.

A method for inducing immunity to a flavivirus in a vertebratesusceptible to infection to the flavivirus is also disclosed. Inparticular, the method is effective for immunizing against infection toa flavivirus having an envelope protein comprising the domain III andcomprises administering an immunogenic effective amount of an aminoacidic molecule comprising a domain III of the envelope protein of theflavivirus. Preferably, the domain III comprises a portion having asequence substantially homologous to SEQ ID NO: 19 or SEQ ID NO: 21.

Accordingly, also a vaccine for flavivirus infection is disclosed,comprising, as an active agent, a polypeptide comprising domain III ofthe envelope protein of the flavivirus. When the flavivirus is West NileVirus, the polypeptide preferably comprise a portion having a sequencesubstantially homologous to SEQ ID NO: 19 or SEQ ID NO: 21.

Also a DNA vaccine comprising as an active agent a vector wherein thedomain III sequence has been disclosed, in particular an adenovirusreplication defective vector is expected to be effective, as well as achimera peptide vaccine against a flavivirus comprising as an activeagent a chemically synthesized peptide of the flavivirus envelope domainIII region, in particular when the flavivirus is West Nile Virus ordengue.

The vaccine may advantageously contain other components, such asadjuvant, attenuated flavivirus, killed flavivirus or subunits thereof,and/or other immunogenic molecules against the flavivirus, in particularagainst the envelope protein of the flavivirus. The manufacturingprocess and components to be included in the vaccine are known as suchto the person skilled in the art and will not be disclosed here indetail.

Also a method of treating a flavivirus infection in a vertebrate in needof such a treatment, in particular humans, is disclosed. This methodcomprises administering to a subject in need of such treatment animmunologically effective dose of the vaccine here disclosed.

The vaccine disclosed herein may be administered by any suitable route,which delivers an immunoprotective amount of the domain III and otherimmunogenic components of the vaccine to the subject. Routes ofadministration of the vaccine, such as, for example, parenteral route,intramuscular route or deep subcutaneous route, are identifiable by aperson skilled in the art. Other modes of administration may also beemployed, where desired, such as oral administration or via otherparenteral routes, i.e., intradermally, intranasally, or intravenously.

A person skilled in the art can determine the appropriateimmunoprotective and non-toxic dose of such vaccine to be administered.The appropriate immunoprotective and non-toxic amount of the activeagents in the vaccine are in the range of the effective amounts ofantigen in conventional vaccines including active agents. The specificdose level for a specific patient will be determined with reference tothe age, sex, and general health of the patient. Also, the synergisticeffect with other drugs administered as well as the diet of the patient,the time and route of administration, and the degree of protection to besought, will be taken in consideration to determine the appropriateimmunoprotective dose for the patient. The administration can berepeated at suitable intervals, if necessary.

A method for treating a flavivirus infection in a vertebrate, when theflavivirus exhibits a flavivirus envelope protein comprising a domainIII is also disclosed. A pharmaceutically effective amount of an agentfunctionally interfering with the domain III of the envelope protein ofthe flavivirus is administered.

Also a pharmaceutical composition is disclosed herein, for treatment ofa flavivirus infection in a vertebrate when the flavivirus exhibits anenvelope protein comprising domain III. The pharmaceutical compositiondisclosed comprises a pharmaceutically effective amount of an agentinterfering with domain III of the envelope protein and apharmaceutically acceptable carrier, vehicle or auxiliary agent.

The agent functionally interfering with domain III of the flavivirus inthe methods of treatment and pharmaceutical composition can be any agentfunctionally interfering with domain III, able to inhibit the entry ofthe flavivirus in the cell herein disclosed. Preferably, the agent is afunctional blocking antibody against domain III, most preferably afunctional blocking polyclonal antibody against domain III, inparticular murine antibodies. Also preferred is a competitive ligand ofdomain III, which can be a competitive natural ligand such as anintegrin including at least one of subunits αV or β3, most preferablythe integrin αVβ3, a neurotensin receptor or an ATPase, preferably aF-ATPase or a V-ATPase. Preferred agents functionally interfering withdomain III, able to inhibit virus entry also include competitivesynthetic ligands, such as chemically synthesized peptides that arecomplementary to domain III or the binding region on the integrin αV β3,neurotensin receptor and/or F-ATPase or V-ATPase.

A pharmaceutically acceptable carrier, vehicle or auxiliary agent asused herein can be identified by a person skilled in the art as suitedto the particular agent and to the particular dosage form desired. Thecomposition may be prepared in various forms for administration,identifiable by a person skilled in the art.

The agent functionally interfering with domain III as described abovemay be administered using any amount and any route of administrationeffective for attenuating infectivity of the virus. Thus, the expression“pharmaceutically effective amount”, as used herein, refers to anontoxic but sufficient amount of the antiviral agent to provide thedesired treatment of viral infection.

The agent described herein may be administered as such, or in the formof a precursor from which the active agent can be derived. Such aprecursor is a derivative of a compound described herein, thepharmacologic action of which results from the conversion by chemical ormetabolic processes in vivo to the active compound. Such a precursor maybe prepared according to procedures well known in the field of medicinalchemistry and pharmaceutical formulation science for each agentdescribed herein.

The administration of the agent functionally interfering with domain IIImay be performed by routes identifiable by the person skilled in the artdepending on the agent administered and the nature and severity of theinfection to be treated.

The exact amount required for the treatment of a subject, and route ofadministration of such amount will vary from subject to subject,depending on the species, age, and general condition of the individualpatient, the severity of the infection, the particular antiviral agentand its mode of administration, etc.

In view of the inhibitory effect on flavivirus infection, the agentinterfering with domain III of the flavivirus envelope protein will beuseful not only for therapeutic treatment of virus infection, but alsofor virus infection prophylaxis.

A method for diagnosing a flavivirus infection in a vertebratesusceptible to infection by the flavivirus is also disclosed. The methodcomprises: contacting a sample tissue from the vertebrate with an agentable to bind domain III of the envelope protein of the virus, inparticular an antibody against domain III, a ligand of domain III or amolecule substantially homologous thereto, associated with anidentifier; and detecting presence or absence of a flavivirus-integrincomplex or flavivirus-neurotensin complex by detecting presence of theidentifier. Alternatively, the identifier can be associated with thesample tissue. Preferably, the antibody against domain III arepolyclonal antibodies, and the ligand is a competitive natural ligandsuch as integrin, a neurotensin receptor protein or an ATPase or acompetitive synthetic ligand.

For example, a plasma membrane of the sample tissue or cell line can beextracted, for example with the protocol given in the reference (Chu andNg, 2003). The extracted plasma membrane can be coated onto 96 wellplates and Cy5-labelled WNV particles can be added for the interaction.After extensive washing to remove background noise, the presence of WNVparticles binding to the receptor molecules can be detected, for exampleby fluorescence in an ELISA plate reader.

A kit for the diagnosis of a flavivirus infection comprising at leastone of the above agents able to bind domain III, optionally associatedor to be associated with an identifier, and one or more reagents able todetect the identifier, is also disclosed, wherein the agent able to binddomain III and the reagents are used according to the diagnostic methodherein disclosed.

The agent able to bind domain III and the one or more reagents able todetect the identifier, can be independently included in one or morecompositions wherein they are comprised together with a suitable vehiclecarrier or auxiliary agent. The identifier can also be included in suchcompositions or in a separate composition to be associated with theagent able to bind the domain or with the cell or sample to be tested.

The identifier and the reagent able to detect the identifier, areidentifiable by a person skilled in the art. Other compositions and/orcomponents that may be suitably included in the kit and are alsoidentifiable by a person skilled in the art.

Also a diagnostic method to detect whether a sample tissue or cell lineis susceptible to flavivirus infection is disclosed, comprisingcontacting a cell with an identifier for the presence or expression ofan integrin, neurotensin receptor and or ATPase to be associated withthe presence or expression of an integrin, neurotensin receptor and orATPase, and detecting the presence of the identifier associated topresence or expression of an integrin, neurotensin receptor and/orATP-ase in the cell.

For example, an approach to detect whether a sample tissue or cell lineis susceptible to flavivirus infection can be that of detecting thepresence or expression of integrin alphaV beta 3 or neurotensin bystaining these cells with antibodies against these receptors and detectfor fluorescence. An exemplary alternative can be to use real-timequantitative PCR to detect the presence of mRNA for integrin orneurotensin receptor in the tissue sample or cells.

A diagnostic kit to detect whether a sample tissue or cell line issusceptible to flavivirus infection is also disclosed, comprising anidentifier for the presence or expression of an integrin, neurotensinreceptor and or ATP-ase, and a reagent able to detect the presence ofthe identifier. The identifier and the reagent able to detect thepresence of the identifier are to be used in the method to detectwhether a sample tissue or cell line is susceptible to flavivirusinfection here disclosed.

The identifier and the reagent can be included in one or morecompositions where the identifier and/or the reagent are included with asuitable vehicle, carrier or auxiliary agent.

In both of the diagnostic kits herein disclosed, the agents andidentifier reagents can be provided in the kits, with suitableinstructions and other necessary reagents, in order to perform themethods here disclosed. The kit will normally contain the compositionsin separate containers. Instructions, for example written or audioinstructions, on paper or electronic support such as tapes or CD-ROMs,for carrying out the assay, will usually be included in the kit. The kitcan also contain, depending on the particular method used, otherpackaged reagents and materials (i.e. wash buffers and the like).

Further details concerning the identification of the suitable carrieragent or auxiliary agent of the compositions, and generallymanufacturing and packaging of the kit, can be identified by the personskilled in the art upon reading of the present disclosure.

Methods, kits, vaccine and pharmaceutical compositions disclosed hereinare particularly used when the flavivirus is a member of the Japaneseencephalitis serocomplex, preferably West Nile Virus, JapaneseEncephalitis virus, West Valley or a virus such as Dengue and Kunjinvirus. Preferably, the vertebrate is a mammal, and, in particular, ahuman being.

A person skilled in the art can identify modalities, dosages, timing ofadministration of the methods herein disclosed as well as vehiclecarrier auxiliary agents, relative concentration, formulation andmodalities of administration of the compositions herein disclosed.

As used herein, the term “antibody” may be a polyclonal or monoclonalantibody unless differently specified. The relevant preparation, isidentifiable by a person skilled in the art upon reading of the presentdisclosure. In the specific examples given, murine polyclonal antibodieswere used. Monoclonal antibodies may be obtained by any technique thatprovides for the production of antibody molecules by continuous cellline culture. These techniques are well known and routinely used inacademic and industrial settings. Some techniques include but are notlimited to the hybridoma technique of Kohler and Milstein, (1975, Nature256:495-497; and U.S. Pat. No. 4,376,110), the human B-cell hybridomatechnique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al.,1983, Proc. Natl Acad. Sci. USA 80:2026-2030), and the EBV-hybridomatechnique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy,Alen R. Liss, Inc., pp. 77-96).

Antibody fragments, which retain the ability to recognize the antigen ofinterest, are included as well.

The antibodies are produced using techniques known to those skilled inthe art and disclosed, for example, in immunization techniques in vivoor in vitro. These techniques are well known and routinely used inacademic and industrial settings.

As used herein, the terms “polypeptide”, and “protein” refer to apolymer of amino acid residues with no limitation concerning a minimumlength of the product. The definition encompasses peptides,oligopeptides, dimers, multimers, and the like, full-length proteins andfragments thereof. The terms also include polypeptides subjected topost-expression modifications such as, for example, glycosylation,acetylation, phosphorylation and the like. Additionally, the term“polypeptide” refers also to a modified protein including proteincomprising deliberate or accidental modifications of the originalsequence, such as deletions, additions and substitutions, so long as theprotein maintains the desired activity.

As used herein, the term “homology” refers to the percent similaritybetween two polynucleotide or two polypeptide moieties. Two DNA, or twopolypeptide, sequences, are “substantially homologous” to each otherwhen the sequences exhibit at least about 50%, preferably at least about70%, more preferably at least about 80%-85%, preferably at least about90%, and most preferably at least about 95%-98% sequence similarity overa defined length of the molecules. As used herein, substantiallyhomologous also refers to sequences showing complete identity to thespecified DNA or polypeptide sequence.

As used herein the term “agent functionally interfering” refers to anykind of interference with the functionality of the molecule, whichresults in a different functionality of the molecule compared with thefunctionality registered in absence of the agent. This includesenhancing and inhibiting the functionality of the molecule. Examples ofinterference with the functionality of the molecule may be obtainedinclude but are not limited to binding the molecule, interference bysteric hindrance with the molecule, modify functional components of themolecule, interfere with the expression of the molecule.

The following examples are provided to describe the invention in furtherdetail. These examples, which set forth a preferred mode presentlycontemplated for carrying out the invention, are intended to illustrateand not to limit the invention.

EXAMPLES

General materials and methods used throughout the experiments will firstbe presented.

Maintenance of Cell Lines

Vero cells (Green monkey kidney) were grown in Medium 199 (M199)containing 10% inactivated fetal calf serum (FCS). Murine neuroblastoma(N2A) cells were maintained in Dulbecco's modified Eagle's mediumsupplemented with 10% FCS. Polarized Vero C1008 cells were grown on 0.4μm porous support membrane insert immersed in M199 supplemented with 10%FCS. The polarity of the cell monolayers was monitored by measuring thetransepithelial electrical resistance with millicell-ERS apparatus. Thenet resistance of the confluent cell monolayers was maintained at 50-70Ω·cm² as calculated based on Blau & Compans (1995). Neurons are isolatedfrom mice and maintained in Dulbecco's modified Eagle's mediumsupplemented with 10% FCS.

Virus Growth & Purification

Vero cells were used to propagate a Flavivirus, West Nile (Sarafend)virus throughout this study. Confluent monolayers of Vero cells wereinfected with West Nile virus at a multiplicity of infection (MOI) of 10PFU/ml. At 24 hr post infection (p.i.), the supernatant was harvested bycentrifugation at 5000 rpm for 10 minutes. West Nile viruses were thenconcentrated and partially purified by centrifugal filter device at 2000rpm for 2 hours. The partially purified viruses were then applied to a 5ml 25% sucrose cushion for further purification. Sucrose gradient wascentrifuged at 25,000 rpm for 2.5 hours at 4° C. in a SW55 rotor.Finally, the purified virus pellet was re-suspended in THE buffer (50 mMTris-HCl, 100 mM NaCl, 1 mM EDTA). The re-suspended virus was aliquoted,snapped frozen and stored at −80° C. The titer of the purified viruspreparation was determined by plaque assay on Vero cells and was foundto be 9×10⁸-5×10⁹ PFU/ml. As a control, supernatant of uninfected Verocells were processed as described above. A similar method was used topropagate Kunjin virus cells, a closely related virus in the samesubgroup, and Dengue virus cells, another flavivirus.

Antibodies and Reagents

The antibody for West Nile virus envelope (E) protein was a monospecificpolyclonal antibody raised in rabbit. The secondary antibody conjugatedto Texas Red (TR) was purchased. Fifteen-nanometer Protein-A colloidalgold was also purchased for antibody detection.

Polyclonal antibodies against the 105 kDa and WNV envelope DIII proteinhave been produced according to the following. The 105-kDa plasmamembrane protein (from Vero cells) or WNV envelope DIII protein wasexcised from the SDS-10% PAGE gels, homogenized, and incubated withImmunEasy mouse adjuvant (Qiagen, USA) at a concentration 3 μg ofprotein per 30 μl of adjuvant. The antigen-adjuvant mixture was used toimmunize BALB/c mice five times subcutaneously at 14-day intervals.Mouse sera were collected 12 days after the last booster. Mouse serawere purified using Econo-Pac serum IgG purification kits (Bio-Rad, USA)and dialyzed overnight with PBS. The purified immunoglobulins werestored at −20° C. Sera were tested by Western blotting detection for thepresence and specificity of antibody against the 105-kDa membraneprotein or DIII protein.

Cell Membrane Protein Preparation

The plasma membrane proteins are prepared as described byMartinez-Barrage and del Angel (2001) and Salas-Benito and del Angel(1997). The integrity of extracted membrane proteins was determined byelectron microscopy as described by Atkinson and Summers (1971). Theconcentration of the protein was determined by Bradford assay withbovine serum albumin (BSA) as the standard. Approximately 800 μg ofproteins were obtained. The membrane protein preparation was aliquotedand stored at −20° C.

Example 1 Protease, Phospholipase, Glycosidase and Lectin Treatment ofCells

To determine the biochemical components (e.g. lipids, proteins orcarbohydrates) of West Nile virus receptor molecules on the surface ofVero cells, cells were pretreated with a panel of enzymes or chemicalsthat would destroy the individual membrane components.

Cell monolayers (Vero cells or N2A) of approximately 5×10⁶ cells werewashed twice with phosphate buffer saline (PBS) before enzyme treatment.Cell monolayers were incubated with the proteases and phospholipases,glycosidases and lectins (as listed below) in PBS at a pH of 7.0 for 45min at 25° C. After treatment, cell monolayers were washed twice withPBS supplemented with 2% FCS to remove the enzymes. Cell monolayers werethen incubated with West Nile virus (MOI=10) at 37° C. for 1 hour.Excess virus particles were inactivated with sodium citrate buffer (pH2.8) for 10 minutes and the cell monolayer washed twice with PBS. Verocells were then incubated at 37° C. for 12 hours. At 12 hours p.i.,virus titers from the treated samples were determined by plaque assays.Three independent experiments were conducted for each set of enzymesused.

The enzymes used were; Proteases: Proteinase K (EC 3.4.21.64) fromTritirachium album, concentration of 10 μg/ml, 1 μg/ml & 0.1 μg/ml;α-chymotrypsin (EC 3.4.21.1) from bovine pancreas, concentration of 10μg/ml & 1 μg/ml; trypsin (EC 3.4.21.4) from bovine pancreas,concentration of 10 μg/ml, 1 μg/ml & 0.1 μg/ml; Bromelain (EC 3.4.22.32)from pineapple stem, concentration of 20 mUnit/ml, 2 mUnit/ml & 0.2mUnit/ml; Papain (EC 3.4.22.2) from Carica papaya, concentration of 50mUnit/ml & 10 mUnit/ml. Phospholipases: Phospholipase A₂ (EC 3.1.1.4)from bovine pancreas, concentration of 1 UNIT/ml & 0.1 UNIT/ml;Phospholipase C (EC 3.1.1.4.3) from Clostridium perfringes,concentration of 10 UNIT/ml & 1 UNIT/ml; Phospholipase D (EC 3.1.4.4)from peanut, concentration of 100 UNIT/ml & 10 UNIT/ml; Glycosidases:Endoglycosidase H (EC 3.2.1.96) from Streptomyces plicatus,concentration of 100 mUNIT/ml & 10 mUNIT/ml; O-glycosidase (EC3.2.1.97)from Diploccus pneumoniae, concentration of 1 mUNIT/ml & 0.1 mUNIT/ml;α-mannosidase (EC 3.2.1.24) from almonds, concentration of 1000 μ/ml,100 μg/ml & 10 μg/ml; α-Fucosidase (EC 3.2.1.11) from almond meal,concentration of 100 mUNIT/ml & 10 mUNIT/ml; Heparinase I (EC 4.2.2.7)and Heparinase III (EC 4.2.2.2.8) from Flavobacterium heparinum,concentration of 1 UNIT/ml & 0.1 UNIT/ml. Lectins: Concanavalin-A fromJack bean, wheat germ agglutinin from Triticum vulgaris,phytohemagglutinin from Phaseolus spp., concentration of 1000 μg/ml &100 μg/ml; Sodium periodate concentration of 1 mM & 0.1 mM. Cellviability after enzyme treatment was assessed by Trypan blue stainingand observation under phase contrast microscope BX 60.

Treatment with glycosidases, sodium periodate and lectins was made toinvestigate possible involvement of carbohydrate moieties on the plasmamembrane for West Nile Virus. In particular, lectins (highly specificcarbohydrate binding molecules) are widely used to determine the natureof carbohydrates involved in ligand-receptor interaction (Liener et al.,1986). Vero cells were then incubated with lectins such as Wheat germagglutinin (which binds to GlcNacβ1-4 on N-linked glycans), concanavalinA (which binds to β3-linked terminal mannose residues on N-linkedglycans) and phytohemagglutinin (which binds oligosaccharides) to assesstheir effects on West Nile virus entry.

The enzymes or chemicals were used at concentrations known to beeffective in reducing the entry of other known viruses (Borrow andOldstone, 1992; Ramos-Castaneda et al., 1997; Salas-Benito and delAngel, 1997; Martinez-Barragan and del Angel, 2001). Results areexpressed as the number of log unit inhibition with respect to untreatedsamples. At the same time, cell viability after treatments was alsoassessed by Trypan blue exclusion method. The number of viable cellsafter treatments was not significantly different from untreated(control) numbers.

FIGS. 1A and 1B show the effects of phospholipases, proteases,glycosidases, sodium periodate and lectins treatments on Vero cells andthe subsequent ability of the cells to allow West Nile virus infection.

In particular, results shown in FIG. 1A, demonstrate that treatment ofVero cells with the three phospholipases does not cause any significantreduction in the productive yields of West Nile virus. Vero cells werealso treated with a panel of proteases which included both serine andthiol proteases. Pretreatment of Vero cells with proteases exhibited adosage-dependent inhibition of West Nile virus entry. Papain, a cysteineendopeptidase that solubilized integral membrane protein, showed thehighest inhibition (approximately a 5-log unit inhibition) of West Nilevirus infection. Therefore, these results show that the cellularreceptor molecule responsible for West Nile virus entry is of aproteineous nature.

In particular, results shown in FIG. 1B demonstrate the effects oftreatment with heparinases, glycosidases and sodium periodate. Withreference to heparinases, pretreatment of a cell with heparinases has noeffect on the entry of West Nile virus. This result was furthersupported by a virus entry blockage study using anti-heparan sulfateproteoglycan treatments of cells.

As per the glycosidases, both Endoglycosidase H and α-mannosidase (whichhydrolyzes N-linked oligossacharides with mannose structures andα-mannose residues respectively) had a significant inhibition on WestNile virus binding and entry into Vero cells. In agreement with thisresult, pretreatment of cells with sodium periodate also substantiallyreduced the binding ability of the cells for West Nile virus. Sodiumperiodate works by oxidizing cell surface carbohydrate residues, butwithout altering protein or lipid epitopes. As for O-glycosidase,α-fucosidase and Heparinase I and III treatments, these enzymes hadminimal effect on West Nile virus infection. In this series ofglycosidase treatments, protease inhibitors cocktails were included toprevent possible contamination by proteases.

With reference to treatment with lectins, blocking of the mannoseresidues on N-linked glycans with concanavalin-A on the cell surfacesprevented the entry of West Nile virus into Vero cells.

Therefore, these preliminary results suggested that the West Nile viruscellular receptor molecule(s) on Vero and N2A cells is a glycoproteinwith complex N-linked sugars containing α-mannose residues.

Example 2 Isolation of Receptor Protein

In these experiments, plasma membrane proteins were isolated and puritywas checked under the electron microscope. The purity of the plasmamembrane extract was considered acceptable with reference to Atkinsonand Summers (1971). Equal quantities of the membrane proteins wereloaded into different gel lanes and separated by SDS-PAGE andtransferred onto nitrocellulose membranes. The nitrocellulose membraneswere incubated sequentially with purified West Nile virus, rabbitpolyclonal mono-specific antibody against the viral envelope protein anddetection by secondary anti-rabbit antibodies conjugated with alkalinephosphatase with addition of substrate (NBT).

In particular, in order to isolate the West Nile virus binding cellreceptor proteins in Vero and N2A plasma membrane extracts, VOPBAs wereperformed. Membrane proteins (80 μg) from either Vero or N2A cells wereseparated by sodium dodecyl sulfate-10% polyacrylamide gelelectrophoresis (SDS-PAGE) as described by Sambrook and co-workers(1989). Proteins separated by SDS-PAGE were electrophoreticallytransferred onto a nitrocellulose membrane using a Western Blottingtransfer apparatus for 3 hours at 4° C. The nitrocellulose membrane wassoaked overnight in a milk buffer (5% skim milk and 0.5% BSA) to blocknon-specific binding sites and to allow re-naturation of the separatedproteins. The membrane was rinsed with PBS (three times) and incubatedsequentially with (a) the purified West Nile virus (prepared as describeabove) for 6 hours at 4° C. (b) mono-specific polyclonal antibodiesagainst West Nile virus envelope protein for 1 hour at 37° C. (c)secondary antibody (anti-rabbit IgG) conjugated with alkalinephosphatase (Chemicon Int, USA) for 45 minutes at 37° C. All incubationswere carried out on a rocking platform and membranes were washed threetimes with a washing buffer (containing 50 mM Tris and 200 mM NaCl with0.05% Tween 20). Non-specific binding of the virus particles was reducedwith a high salt buffer wash. The presence of virus binding was detectedby the addition of substrate, nitroblue tetrazolium. Finally, themembranes were washed with distilled water and dried (and/or put instripping and re-probing protocol).

A 105-KDa band was detected. The corresponding 105-kDa plasma membraneprotein (from Vero cells) was excised from the SDS-10% PAGE gels,homogenized and incubated with ImmunEasy mouse adjuvant at aconcentration recommended by the manufacturer, Qiagen. Theantigen-adjuvant mixture was used to immunize BALB/C mice five timessubcutaneously at 14-day intervals. Mouse sera were collected 12 daysafter the last booster. Mouse sera were purified using Econo-Pac serumIgG purification kits and dialyzed overnight with PBS. The purifiedimmunoglobulins were stored at −20° C. Sera were tested by Westernblotting detection for the presence and specificity of antibody againstthe 105-kDa cell receptor protein.

Results are shown in FIGS. 2A and 2B. West Nile virus was observed tobind to a 105-kDa band in the membrane preparations of both Vero and N2Acells (FIGS. 2A and 2B, respectively, Lane 1). No bands were observedwhen supernatants of uninfected cells (prepared as according to thesupernatants of West Nile virus-infected cells) were incubated under thesame conditions (FIGS. 2A and 2B, respectively, Lane 2).

To ensure this was a specific interaction between the virus and the105-kDa cell receptor protein, several procedures were carried out. Inparticular, despite high salt (200 mM NaCl) and detergent (0.05% Tween20) washing, West Nile virus still binds strongly to the 105-kDa cellreceptor proteins.

Example 3 Protease Treatment of Membrane Proteins

To affirm the results of enzymes and chemical treatments of intact Verocells reported in the previous examples, VOPBAs were also performed onplasma membrane protein extracts that were treated with protease(papain).

After papain treatment of both vero and N2A cells, the membrane proteinswere isolated and prepared and VOPBAs were performed according to theprocedure described in example 2.

The results are shown in FIG. 3. WN virus binding to the membraneproteins of both Vero and N2A cells after papain treatment at theconcentration of 50 mUnit/ml is abolished (Lanes 2 & 4 respectively) ascompared to that of the untreated cells (Lanes 1 & 3 respectively).These results confirm that the receptor is of a proteinaceous nature.Additionally, since also after this treatment a 105 KDa band wasobserved, the existence of a 105-KDa virus receptor was furtherconfirmed.

Example 4 Kinetics of West Nile Virus Binding Molecules

Following the experiments reported in example 3, the kinetics of WestNile virus binding molecules returning to the cell surface after removalwith papain was also examined.

Vero cells (approximately 5×10⁶ cells) were treated with papain at 50mUnit/ml in PBS or PBS (untreated) for 45 minutes at 25° C. Another setof Vero cells was incubated with 100 μg/ml of cycloheximide to block newprotein synthesis, for 2 hours prior to papain treatments. Treated cellswere washed twice with PBS supplemented with 2% FCS to inactivate theenzyme. Fresh M199 plus 10% FCS with or without cycloheximide (100μg/ml) were added to the cells. The cells were then incubated at 37° C.in 5% CO₂. At specific times after incubation (0, 2 & 4 hours), plasmamembrane proteins were extracted as herein described and VOPBA wasperformed.

The results of these experiments are shown in FIG. 4. Vero cells werefirst treated with papain (50 mU/ml) for 45 minutes at room temperatureand after protease (papain) removal after 0, 2 and 4 hours, virusbinding was determined by VOPBA. No virus binding was observed at 0 hrfollowing the removal of the protease [FIG. 4—Lane 2 (withoutcycloheximide and Lane 5 (with cycloheximide)] when compared to themembrane protein that is not treated with papain (Lane 1). West Nilevirus binding to the 105-kDa cell receptor protein was observed after 2hours with the removal of the protease and reached its original levelwithin 4 hours (FIG. 4—Lanes 3 & 4 respectively). Despite the blockageof new protein synthesis by cycloheximide, virus binding was alsoobserved after 2 hours and 4 hours (FIG. 4—Lanes 6 & 7 respectively).This indicates the presence of abundant pre-existing internal pools ofthe 105-kDa cell receptor proteins that were rapidly trafficked to thecell surface after removal without the need for new protein synthesis tooccur.

Example 5 Glycosidase Periodate and Lectins Treatement of MembraneProteins

Carbohydrate residues on cell surfaces have been shown to be importantfor the initial binding of viruses, which would then mediate thesubsequent entry of the virus through its high affinity receptor. Thenature and roles of carbohydrate residues present on the 105-kDa proteinfor West Nile virus binding were further assessed by VOPBA.

In particular, VOPBAs were also performed on plasma membrane proteinextracts (obtained with the procedure reported in example 4) that weretreated with glycosidases (Endoglycosidase H, α-mannosidase andO-glycosidase), sodium periodate and lectins.

After each treatment, membrane proteins were isolated and prepared, andVOPBAs performed according to the procedures described in Example 2.

Results of these experiments are shown in FIGS. 5A to 5C. FIG. 5A showsthe results of glycosidase treatments of membrane proteins. No bindingof West Nile virus was observed after treatment with α-mannosidase andEndoglycosidase H in both Vero (Lanes 2 and 3) and N2A (Lanes 6 and 7)plasma membrane protein extracts, when compared with the untreatedmembrane proteins (Vero cells—Lane 1 and N2A cells—Lane 5). In contrast,O-glycosidase treatment of the membrane proteins (Vero cells—Lane 4 andN2A cells—Lane 8) did not affect the binding of West Nile virus to the105-kDa protein band. It could be deduced that virus binding to the105-kDa cell receptor protein is neither mediated by O-linked sugars norcontains O-linked glycoslation. The nature of carbohydrates present inthe 105-kDa cell receptor proteins necessary for West Nile virus bindingwas further assessed by lectin treatments using VOPBA.

Based on the results shown in FIG. 5B, concananvalin-A was observed toblock the binding of West Nile virus to the 105-kDa cell receptorproteins in a dosage-dependent manner [Lane 1 (untreated), Lane 2 (10μg/ml) and Lane 3 (100 μg/ml)]. On the other hand, phytohemagglutininhad no effect in blocking virus binding to the cell receptor proteins[equal intensities—Lane 4 (untreated), Lane 5 (10 μg/ml) and Lane 6 100μg/ml)].

Similarly, FIG. 5C shows that binding of West Nile virus to the sodiumperiodate-treated membrane proteins was reduced in a dosage-dependentmanner (FIG. 5C, Lane 1—untreated, Lane 2—0.1 mM, Lane 3—1 mM and Lane4—10 mM). These results have provided more evidence that the 105-kDacell receptor protein contains carbohydrate groups with high mannoseresidues that are important for virus binding.

Therefore, treatment of membrane proteins with Endoglycosidase H orα-mannosidase abolished virus binding (FIG. 5A) while sodium periodateexhibited a dosage dependent inhibition of West Nile virus binding tothe 105-kDa cell receptor protein (FIG. 5C). Since Endoglycosidase Hcleaves only the high mannose residues of N-linked oligosaccharides onglycoproteins and concanavanlin-A binds specifically to mannose residues(FIG. 5B), this further emphasizes importance of N-linked sugars withmannose residues on the 105-kDa cell receptor protein for West Nilevirus binding.

Example 6 β-Mercaptoethanol Treatment of Membrane Proteins

To investigate the possible presence of di-sulfide-linked bridges in the105-KDa plasma protein, plasma membrane extracts according to theprocedure reported in example 4, were also treated with 5 mM ofβ-mercaptoethanol.

Interestingly, a faint 105-kDa band and a series of protein bandsranging from 30 to 40-kDa were observed after treatment withβ-mercaptoenthanol, followed by VOPBA [FIG. 6—Lanes 2 (Vero cells) & 4(N2A cells)]. Lanes 1 and 3 are untreated samples from Vero and N2Acells, respectively. These results may suggest that treatment withβ-mercaptoethanol did not disrupt virus binding and virus binding occursmainly through the interaction with the carbohydrate moieties instead ofthe peptide portion of the glycoprotein.

Consistent with treatment of the membrane proteins withβ-mecaptoethanol, West Nile virus was observed to bind to a series ofprotein bands ranging from 30 to 40-kDa (FIG. 6). This may indicate thatthe 105-kDa cell receptor protein is made up of di-sulfide linkedsubunits. However, it might be equally plausible that the virus bindingprotein is actually 30 kDa, and upon cell lysis it becomes cross-linkedvia inadvertent di-suflide linkage to other proteins.

To investigate the actual molecular weight of the virus binding protein,the plasma membrane extraction procedure was repeated in the presence ofalkaylating agent (iodoacetamide) to block thiol reactivities.Consistent with the result obtained in the absence of alkaylating agent,West Nile virus binds to a single 105-kDa protein.

Furthermore, the action of β-mecaptoethanol did not seem to affect virusbinding to the protein subunits. This interesting result suggests eitherthat West Nile virus binding did not require a folding dependentdi-sulfide bridge or that the West Nile virus binds to the carbohydratesresidues on the protein not affected by the action of β-mecaptoethanol.The latter is in line with the above observations that the carbohydrateresidues on the membrane protein are necessary for virus binding.

Example 7 Western Blot Analysis

For analysis of the specificity of the anti-105-kDa polyclonalantibodies, plasma membrane proteins from Vero cells were separatedusing SDS-polyacrylamide gel electrophoresis and transferred tonitrocellulose membrane (Biorad, USA). The Western blot procedure wascarried out as described in Chu and Ng (2002). The blot was thenincubated overnight in the anti-105-kDa protein antibodies or pre-immuneserum at room temperature on an orbital shaker for 1 hour. Reactionswere then detected by staining with alkaline-phophatase conjugated goatanti-mouse IgG (Chemicon Int, USA) with the addition of substrate,nitroblue tetrazolium (NBT).

Results are shown in FIG. 7. The murine polyclonal antibody generatedagainst the 105-kDa protein was shown to be specific for the 105-kDaprotein (from Vero cells) by Western blot assay (FIG. 7, Lane 2) whileno bands were observed after incubation with preimmune serum (FIG. 7,Lane 1). This murine polyclonal antibody was also specific for the105-kDa cell receptor protein from N2A cells.

Example 8 Indirect Immunofluorescence Confocal Microscopy

To determine the localization of the 105-kDa proteins on Vero cells,immunofluorescence assays were carried out. To determine whether the105-kDa cell receptor protein is differentially expressed in polarizedcells, immunofluorescence assays coupled with optical sectioning bylaser scanning confocal microscopy were also carried out with Vero C1008cells. The Vero C1008 is a polarized cell line derived from Vero cells.These epithelial cells have distinct apical and basolateral domains ofthe plasma membrane.

For immunofluorescence microscopy, cell monolayers were grown on coverslips or 0.4 μm porous support membrane inserts. The subsequentprocedure is similar to that described in Chu & Ng (2002). The primaryantibody used was anti-105-kDa polyclonal antibody (with a 1:100) andthe fluorochrome were Texas Red (TR)-conjugated secondary antibodies.The specimens were viewed with laser scanning confocal invertedmicroscope (excitation wavelength of 543 nm for TR) using oil immersionobjectives.

Results are shown in FIG. 8A. The 105-kDa cell receptor protein wasmainly localized to the plasma membrane as detected by the anti-105-kDaprotein antibodies using indirect immunofluorescence. This is a typicallocalization pattern for cell surface molecules. Despite methanolfixation and permeabilization, the murine polyclonal antibodies againstthe 105-kDa protein still bind specifically to the plasma membrane(arrows).

Results of experiments performed in polarized cells are shown in FIG.8B. In polarized epithelial cells (Vero C1008), there was a high levelof expression of the 105-kDa proteins at the apical surface as comparedto the basolateral surface (FIG. 8B). This result could explain for thepreferential entry of West Nile virus through the apical surface of thepolarized Vero C1008 cells as illustrated in a previous study (Chu andNg, 2002).

Example 9 Cryo-Immunolabelling Electron Microscopy

To confirm the results of example 8, cryo-immuno-labelling electronmicroscopy also was carried out.

Vero cells were incubated with West Nile virus (MOI=100) at 4° C. for 30minutes to allow virus attachment to the plasma membrane. The cells werethen processed for cryo-electron microscopy using the Tokuyasu method(1984) with some modifications as described in Ng and colleagues (2001).Briefly, the cells were fixed in 4% paraformaldehyde and 0.2%glutaldehyde followed by embedding in gelatin. The gelatin block withthe cells was immersed in cryo-protectant, rapidly frozen beforecryo-ultramicroscopy, using an ultramicrotome (UCT) having acryo-attachment.

For immuno-labeling, the primary antibody was the anti-105-kDa membraneprotein (1:100 dilution) followed by conjugation with Protein Acolloidal gold (at dilution 1:20). The sections were viewed under theCM120 Biotwin transmission electron microscope.

Results are shown in FIG. 9. In particular, localization of the 105-kDaprotein was confirmed by the immuno-gold labeling of cryo-sections. Moreparticularly, at the ultrastructural level, West Nile virus was observedto bind to the 105-kDa cell receptor proteins as defined by the 10 nmgold particles—(arrows) at the plasma membrane. The use of immuno-cryoelectron microscopy revealed the specific binding of West Nile virus tothe 105-kDa cell receptor protein.

Example 10 Inhibition of Binding of West Nile Virus to Membrane Cells byReceptor Protein Polyclonal Antibodies

Inhibition of binding of West Nile virus to membrane cells by 105-KDaprotein was tested.

After pre-incubation with the 105 KDa protein antibodies, membrane cellsproteins were isolated and prepared, and VOPBAs performed according tothe procedure reported in example 2.

Results are shown in FIG. 10. The pre-incubation of the 105 kDa proteinantibodies on the separated membrane proteins through VOPBA alsoprevented the binding of West Nile virus (FIG. 10, Lane 1). Virusbinding occurred in the absence of the 105-kDa protein antibodies (Lane2). Hence, these results provide strong evidence that the 105-kDa cellreceptor protein is a possible cellular receptor for West Nile virus andother closely-related flaviviruses.

Example 11 Inhibition of West Nile Virus Infection by Receptor ProteinPolyclonal Antibodies

This set of experiments was carried out to determine if the antibodiesagainst the 105-kDa protein recognized the same cell receptor proteinfor West Nile virus entry. Blockage of West Nile virus cellularreceptors with specific antibodies against the cell receptor proteinwould prevent virus entry.

Confluent monolayer of Vero cells were first washed twice with PBS andpreincubated with preimmune serum or the anti-105-kDa polyclonalantibodies for 1 hour at 37° C. After incubation, cells were washedthrice with PBS and infected with West Nile virus, Kunjin virus (aflavivirus in the same subgroup as West Nile virus), Dengue (anotherflavivirus) (MOI=10). At appropriate p.i. time periods, supernatantsfrom the virus-infected cells were processed for plaque assays. Forcontrol purposes, the above procedures were repeated with an unrelatedpoliovirus infection.

Results are shown in Table 1 below.

TABLE 1 Log unit inhibition of infectivity by pre- immune sera at thefollowing dilutions. 1:10 1:100 1:1000 WN 0.25 ± 0.56 0.15 ± 0.85 0.20 ±0.50 Kunjin 0.35 ± 0.22 0.28 ± 0.60 0.32 ± 0.68 Polio 0.95 ± 0.25 0.89 ±0.87 0.88 ± 0.75 Log unit inhibition of infectivity by anti-105 kDaMembrane Protein at the following dilutions. 1:10 1:100 1:1000 WN  6.5 ±0.80  4.5 ± 0.45  0.6 ± 0.50 Kunjin  4.0 ± 0.35  2.8 ± 0.60 0.35 ± 0.20Polio 0.85 ± 0.90 0.90 ± 0.55 0.75 ± 0.20

The entry of West Nile and kunjin virus was strongly inhibited, whilepreimmune sera did not cause any inhibition. In contrast, the entry ofpoliovirus, a non-related picornavirus, was not affected in the presenceof the 105-kDa protein antibody.

These results therefore strongly support the conclusion that the 105-kDaglycoprotein is the receptor for West Nile virus. Additionally, sincethe anti-105-kDa membrane protein antibodies were also effective inblocking the entry of the flavivirus kunjin virus, other flaviviruses ofthe Japanese encephalitis serocomplex subgroup, such as St. Louisencephalitis, Murray Valley encephalitis, kunjin viruses, dengue virus1, dengue virus 2, dengue virus 3 and dengue virus 4, might also utilizethis 105-kDa cell receptor protein for entry into host cells.

Example 12 Location of the 105-kDa Cell Receptor Protein in Vertebratesand Organs

Detection of the cell receptor protein from plasma membrane extractsfrom cells of several species, including vertebrates, utilizing a WestNile virus antibody and/or the cell receptor protein antibody isexpected. Exemplary species comprise crows, horses, mice and humans, todetermine if the cell receptor protein is present in these groups and ifit can bind a flavivirus. This is shown by providing membrane proteins,loaded into different gel lanes, separated by SDS-PAGE and transferredonto nitrocellulose membrane. The nitrocellulose membranes are incubatedsequentially with purified West Nile virus, rabbit polyclonalmono-specific antibody against the viral envelope protein and thendetected by secondary anti-rabbit antibodies conjugated with alkalinephosphatase with addition of substrate (NBT), for example.

The nitrocellulose membranes can be stripped and re-probed with the 105kDa protein antibody. Exemplary incubation of separated membraneproteins with preimmune serum and antibodies at a dilution of 1:500 forcrows, horses, mice and humans is contemplated, as well as use of Goatanti-mouse IgG conjugated with alkaline phosphatase, at a dilution of1:2000. The antibody binding generated will be highly specific for105-kDa membrane protein in a range of species. Antibody binding foundin all species indicates that the cell receptor protein is present in awide range of vertebrates. This would account for the wide pathogenicityof the West Nile virus among vertebrates.

Example 13 Peptide Sequencing of the 105-kDa Cell Receptor Protein

Peptide sequencing of the 105 KDa membrane-associated glycoprotein wascarried out to determine the identity of the glycoprotein following theprocedure disclosed in Sagara et al, 1998

The amino acidic sequences reported in the sequence listing as SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, havebeen obtained as a result. These sequences closely match to those of theintegrin superfamily after performing a database protein homology searchin the databases Entrez Protein and NCBI, following procedures known tothe person skilled in the art. Therefore, the peptide sequencing showsthat the 105 KDa glycoprotein belongs to the integrin superfamily.

Example 14 105-kDa Cell Receptor Protein Identity

To determine the specific integrin molecule(s) or its (their) subunit ofthe integrin superfamily that mediate binding and entry of West NileVirus, Vero cells were pre-incubated with a panel of functional blockingantibodies against integrins (α1, α2, α3, α4, α5, α6, αV, β1, β2, β3,β4, β5-Chemicon USA and Santa Cruz Biotech, USA) following proceduresdisclosed in Sagara et al 1998 at 4° C. (to determine virus binding) or37° C. (to determine virus entry). Antibodies from different Inparticula antibodies from different companies has been used to ensurereliability of results on inhibitory effect on West Nile virus bindingto Vero cells.

Radioactive labeled West Nile Virus were then added to the treated cellsand further incubated for 1 h at 4° C. or 37° C. Excess and unboundparticles were inactivated in acid glycine buffer (pH3) and removed bywashing with PBS for three times. Results concerning virus binding areshown in FIG. 11A. Results concerning virus entry are shown in FIG. 11B. A significant inhibition of the entry of the virus is shown bytreatment of antibodies against all the integrins subunits tested.Antibodies against integrin αVβ3 and its individual subunits (αV and β3)showed the highest inhibition of West Nile virus binding and entry. AntiαVβ5 integrin antibodies also show some extent of West Nile virusbinding and entry. In particular αVβ5 is also capable of blocking thebinding of West Nile virus and much lesser effect on the entry of WestNile Virus, showing that integrin alpha V is functioning as the specificbinding molecule for WNV.

This experiment is repeated with another closely related flavivirus,Japanese Encephalitis Virus using all the anti integrin antibodies at 25μg/ml. Results are shown in FIG. 12. Antibodies against integrinsubunits (αV and β3) and integrin αVβ3 strongly inhibit the entry ofJapanese Encephalitis Virus into Vero cells.

Therefore, the results obtained show that αVβ3 is the receptor moleculefor both West Nile and Japanese Encephalitis virus and that both theintegrin subunits of αV and β3 are required in binding to West Nile andJapanese Encephalitis virus.

Example 15 Role of Divalent Cations in the Binding of WNV and JEV

Confluent Vero cells were first washed with phosphate buffered saline(pH 7.4) and added with fresh culture medium M199 with 1% FCS containingEDTA (3-12 mM). The cells were incubated with EDTA for 2 hrs at 37° C.After the incubation period, Vero cells were added with radiolabelledWNV particles and assess for its entry into Vero cells.

Divalent cations (Ca²+) have been shown to be involved in the specificbinding of physiological ligands to integrin. The possible requirementof divalent cations for binding of WNV to integrins was investigated byusing EDTA (divalent cations chelators). Vero cells were treated withEDTA at a series of concentration that has been shown to inhibit thebinding of physiological ligands to integrin. Results are shown in FIG.13. Vero cells treated with EDTA did not block the binding andsubsequent entry of West Nile virus. Therefore, removal of divalentcations from the culture environment did not have any effect on theentry of WNV into Vero cells.

Example 16 Competitive Physiological Ligand Binding Assay

Competitive physiological ligand binding assay with fibronectin,vitronectin, heparin, chrondriotin sulphate, laminin and the peptidesRGD1 and RGE1 reported in the sequence listing as SEQ ID NO: 6 and SEQID NO:7 was carried out. In particular Vero cells were incubated withdifferent concentrations of ECM proteins, RGD peptides, or anti-integrinantibodies at 4° C. or 37° C. for 1 hr, washed, incubated with 50 μl ofradiolabeled WNV, JEV at 37° C. for 1 hr and assessed for virus entry.

Results are shown in FIG. 14. Binding of fibronectin vitronectin,laminin, and RGD peptide to cell surface has results only in partialinhibition of west Nile virus infection. At the same time, the partialinhibition by RGD peptide and physiological ligand (vitronectin) forintegrin αVβ3 may propose that the binding of WNV to integrin may not behighly dependent on the usage of RGD motif on envelope protein of virus.

Previous studies have shown that many ligands and viruses can bind tointegrin independently in the absence of RGD motif. Furthermore,site-directed mutagenesis of RGD motif in Murray Valley virus(flavivirus-JE serogroup) also shows consistent results that binding ofMurray Valley virus to cells is independent of RGD motif on its envelopeprotein.

Example 17 Distribution and Localization of the 105 KDa Glycoprotein

To compare and confirm the results of experiments reported in examples14 to 16, cell surface staining by immunofluorescence assay was carriedout with antibodies against 105 KDa membrane protein and integrin αVβ3,respectively. Vero cells were fixed with methanol and processed forimmunofluorescence staining. For immunofluorescence microscopy, cellmonolayers were grown on cover slips and fixed with cold absolutemethanol for 10 min. Subsequent procedure is similar to that describedin Chu & Ng (2002). Antibodies against integrin αVβ3 and 105 KDa plasmamembrane glycoprotein were used as the primary antibodies, respectively.Primary antibodies and specific secondary antibodies conjugated withFITC were added subsequently. The primary antibody utilized was ananti-105-kDa polyclonal antibody (1:100) and anti-integrin alphaV beta 3monoclonal antibody (1:500), at indicated dilutions. The fluorochromeused was an FITC conjugated secondary antibody. The specimens wereviewed with Olympus IX81 using oil immersion objectives.

Results are shown in FIGS. 15 A and B, respectively. In particular,integrins αVβ3 are distributed along the plasma membrane and focaladhesion through the cytoplasm (see FIG. 15A). Similar distributionpatterns of the 105 KDa glycoprotein were also observed when compared tointegrin αVβ3 shown in FIG. 15A. Thus, a similar distribution patternbetween the 105 KDa glycoprotein and integrin αVβ3 is observed.

Therefore, these data show that the WNV virus binding 105 KDa plasmamembrane protein is the integrin αVβ3.

Example 18 Gene Knockout and Down Regulation of Integrin αVβ3 in VeroCells

To further confirm that integrin subunits αV and β3 are the receptormolecules for West Nile Virus, gene knockout by means of RNA inteferingwas carried out. Ten short gene sequences from the full length integrinαV gene sequence (SEQ ID NO: 8) and twelve short gene sequences from thefull length integrin β3 gene sequence (SEQ ID NO:10) were selected andligated into BamHI and HindIII digested pSilencer 3.0. In particular thefollowing integrin sequences reported on the sequence listing as fromSEQ ID NO: 12 to SEQ ID NO 15, were used: Integrin alpha V1 (SEQ ID NO:12), Integrin alpha V2: (SEQ ID NO: 13); Integrin beta 31 (SEQ ID NO:14); Integrin beta 32: (SEQ ID NO: 15). All twenty-two clones wereselected and sequenced to verify in-frame insertion. All clones werethen transfected into Vero cells and screened for down regulation ofintegrin expression.

In particular, plasmid constructs (psilencer 3.0-H1, Ambion, USA)containing different regions of the integrin alpha V beta 3 subunits(shown below) were constructed. Transfections were performed usingLipofectamine PLUS reagents from Invitrogen (USA) as specified by themanufacturer. In brief, Vero cells were grown on coverslips in 24-wellstissue culture plate until 75% confluency. 1 to 5 μg of the respectiveconstructs was complexed with 4 μl of PLUS reagent in 25 μl of OPTI-MEMmedium (GIBCO) for 15 min at room temperature. The mixture was thenadded to 25 μl of OPTI-MEM containing 2 μl of lipofectamine. Afterincubation for another 15 min, the DNA-liposome complexes were added tothe cells. Following incubation for 3 h at 37° C., 1 ml of completegrowth medium was added and incubated for another 24 hrs before virusentry assay was carried out. The down regulation of integrin was checkedby immunofluorescence assay using antibodies against integrin alphaV andbeta 3.

The transfection efficiency was determined to be approximately 35%.Results are shown in FIG. 16. Control formed by immunofluorescencestaining of integrin αV and β3 on Vero cells using anti integrin αV andβ3 antibodies is shown in FIGS. 16A and 16C. Those Figures respectivelyshow that integrins αV and β3 are both mainly distributed at the cellsurface and the focal adhesion junction. Vero cells transfected withpSilencer-siRNA integrin αV or integrin β3 shown in FIG. 16B and FIG.16D respectively, show down regulation of integrin αV and β3 on Verocells.

In particular, a number of these clones can strongly down-regulate theexpression of integrin αV or β3 by 80%. These integrin down-regulatedclones were selected for WNV virus entry study. Results are shown inFIG. 17. The down-regulation of either integrin αV or β3 stronglyinhibited the entry of West Nile Virus as compared to internal controlof GADPH.

Example 19 ATPases Antibody Blocking Virus Entry Assays

Antibody Blocking Virus Entry Assays (ABVEA) performed using antibodiesagainst various plasma membrane-associated proteins have shownsignificant inhibition of virus entry of blockage of ATPases (data notshown). To investigate the role of an ATPase as co-receptor of the WestNile Virus, a further series of ABVEA was performed using antibodiesraised against plasma membrane related ATPases.

In particular Vero cells were grown in 96 wells microtitre plates tillconfluent. Cells were washed thrice with PBS and incubated withantibodies against ATPase beta subunit, ATPase alpha subunit, calciumdihydropyridine receptor alpha, calcium dihydropyridine receptor beta,VATPase E and VATPase for 1 hr at 37° C. Excess antibodies were removedby washing thrice with PBS. 50 μl of radiolabeled WNV were added andincubated for another 1 hr at 37° C. Excess virus were then inactivatedand washed with acid glycine buffer (pH 2.8). Penetrated virus were thendetermined.

Results are shown in FIG. 18. The blocking of both plasma membraneassociated ATPases and vacuolar ATPases with their respective antibodiesseem to exert an inhibitory effect on the entry process of WNV. Ingeneral, ATPases are required to generate energy for many cellularactivities across the plasma membrane. Hence, ATPases may act asco-receptor for WNV binding and providing the necessary energy for theendocytosis process of WNV.

Example 20 Neurotensin Antibody Blocking Virus Entry Assays

By using human brain cDNA library screening for interacting partnerswith WNV envelope protein in Yeast-2 hybrid system (not shown), aneurotensin receptor was obtained after several rounds of stringentselection. DNA sequence coding for neurotensin receptor is reported inthe sequence listing as SEQ ID NO: 16, the amino acid sequence reportedas SEQ ID NO:17.

ABVEA were performed with antibodies against the neurotensin receptor inA172 neuroblastoma cells and in Vero cells as a control.

In particular Vero cells were grown in 96 wells microtitre plates tillconfluent. Cells were washed thrice with PBS and incubated withantibodies against nerotensin receptor for 1 hr at 37° C. Excessantibodies were removed by washing thrice with PBS. 50 μl ofradiolabeled WNV were added and incubated for another 1 hr at 37° C.Excess virus were then inactivated and washed with acid glycine buffer(pH 2.8). Penetrated virus were then determined.

Results are shown in FIG. 19. Both A172 and Vero cells werepre-incubated with different concentrations of antibodies againstneurotensin receptors and followed by incubation with radiolabeled WNV.Entry of WNV is significantly inhibited by anti-neurotensin receptorantibodies in A172 cells but not in Vero cells.

These results have been confirmed by WNV competitive binding assays ofneurotensin receptor with its natural ligand. Results of such assays areshown in FIG. 20. Neurotensin (natural ligand) competitively blockedentry of WNV into A172 cells. A172 cells were pretreated withneurotensin at a different concentration before incubation of cells withreadiolabeled WNV. Entry of WNV is blocked in a dosage dependent manner.

Example 21 Effect Of Neurotensin Down Regulation to West Nile VirusEntry in A172 Cells

Work has been carried out to knockout the expression of neurotensinreceptor in A172 cells and assess for West Nile virus entry. Three shortgene sequences from the full length neurotensin receptor sequence wereselected and ligated in BamHI and HindII digested pSilencer 3.0 (notshown). In particular the sequence reported in the sequence listing asSEQ ID NO: 18 was used.

Plasmid constructs (psilencer 3.0-H1, Ambion, USA) containing theneurotensin receptor sequence reported in the sequence listing as SEQ IDNO: 18 was transfected into A172 cells. Transfections were performedusing Lipofectamine PLUS reagents from Invitrogen (USA) as specified bythe manufacturer. In brief, Vero cells were grown on coverslips in24-wells tissue culture plate until 75% confluency. 1 to 5 μg of therespective constructs was complexed with 4 μl of PLUS reagent in 25 μlof OPTI-MEM medium (GIBCO) for 15 min at room temperature. The mixturewas then added to 25 μl of OPTI-MEM containing 2 μl of lipofectamine.After incubation for another 15 min, the DNA-liposome complexes wereadded to the cells. Following incubation for 3 h at 37° C., 1 ml ofcomplete growth medium was added and incubated for another 24 hrs beforevirus entry assay was carried out.

Clones were selected and sequenced to verify in frame insertion (notshown). All clones were then transfected into A172 cells and screenedfor down-regulation of neurotensin receptor expression. The downregulation of neurotensin receptor was checked by immunofluorescenceassay using antibodies against neurotensin receptor. To demonstrateexpression and localization of the receptor immunofluorescence assaywith the antineurotensin receptor, antibodies were used. Results areshown in FIG. 21. A172 cells transfected with pSilencer-siRNA expressingsiRNA against the neurotensin receptor showed a down regulation ofplasma membrane neurotensin receptor expression (B). No down regulationwas observed instead in the control, where in absence of transfectionwith pSilencer-siRNA neurotensin receptor is expressed predominantly onthe plasma membrane and within cytoplasmic vesicles (A).

Example 22 West Nile Virus Attachment Domain

West Nile virus envelope domain III (350-390) was cloned into E. coliexpression vector pET16b (Novogen, USA) and expressed as His-taggedfusion protein. The DIII protein was expressed as a soluble protein andwas purified through a nickel column. The purified DIII protein wasseparated by 10% SDS PAGE and followed by transferring to nitrocellulosemembrane. The recombinant DIII is detected with monoclonal antibodiesagainst E protein of WNV and anti-His antibodies. The monoclonal anti-Eprotein and anti-His antibodies were used at a concentration of 1:500and 1:200 respectively. The secondary antibodies conjugated withalkaline phosphatase were added subsequently. Detection of DIII proteinwas carried out by adding the substrate (nitroblue tetrazolium) to theblot.

Vero cells were first incubated with different concentration (5 to 100μg/ml) of DIII protein or BSA for 30 min at 37° C. Excess or unboundprotein is removed by washing thrice with PBS. Radiolabeled WNV orDengue virus (250 plaque forming unit, PFU) is added and incubated for 1h at 37° C. Virus entry into Vero cells was determined by radioactivecounts from a scintillation counter.

The production of murine polyclonal antibodies against West Nile virusDIII protein was carried out as previously described by Chu and Ng,2003. The pool sera from 6 DIII protein immunized Balb/c mice werediluted in a series of concentration 1:2 to 1:8192. Equal volume (50 μl)of anti-DIII antibodies and WNV (500 PFU) were incubated for 1 h beforeoverlaying onto Vero cells monolayer. Excess or unbound virus-antibodycomplexes were removed by washing thrice with PBS. Plaques were stainedwith crystal violet after 4 days of incubation at 37° C. Virus diluentwas used as a control for anti-DIII antibodies.

The portion of West Nile Virus coding for the domain III is reportedherein as SEQ ID NO: 20. This sequence encodes for domain III of theEnvelope protein according to the well known genetic code. A personskilled in the art can easily identify the domain III coding portionsinside the sequence as well as the amino acid sequence, as encoded bySEQ ID NO: 21.

The amino acid sequence of domain III coded by the sequence reported asSEQ ID NO: 20 is designated as SEQ ID NO: 21. A soluble form ofrecombinant domain III from 350 to 390 of the envelope protein of WestNile virus envelope protein (E protein) was cloned into E. coliexpression vector pET16b (Novogen, USA) and expressed as His-taggedfusion protein. The protein had comprised a sequence reported in thesequence listing as SEQ ID NO: 19. The DIII protein was able to beexpressed as a soluble protein. Recombinant DIII protein was separatedby 10% SDS. PAGE and followed by transferring to nitrocellulosemembrane. The recombinant DIII is detected with monoclonal antibodiesagainst E protein of WNV and anti-His antibodies. The results are shownin FIG. 22.

Also polyclonal antibodies (not shown) against West Nile virus E-proteinwere able to detect the expressed recombinant E domain III (FIG. 22).

Subsequently, Vero cells were first incubated with differentconcentration of DIII protein or BSA. Radiolabeled WNV or Dengue virusis added and assay for virus entry. Results are shown in FIG. 23. Entryof WNV is significantly blocked in the presence of DIII protein whileBSA did not have any effect on the entry of WNV. Recombinant WNVenvelope DIII protein can also slightly block the entry of Dengue virusat high concentration used. Therefore, recombinant E domain III was alsoable to competitively inhibit the binding of West Nile virus in a dosagedependent manner.

In addition, murine polyclonal antibodies were produced against therecombinant domain III (FIG. 24). A single 13 KDA protein band (DIIIprotein) was detected by the murine polyclonal antibodies.

These murine polyclonal antibodies were used in plaque neutralizationassay of WNV. Recombinant DIII is expressed, purified and injected into6 Balb/c mice. Pool sera were obtained and diluted in a series ofconcentration as shown in the graph below. Equal volume of anti-DIIIantibodies and WNV (500 PFU) were incubated for 1 h before overlayingonto Vero cells monolayer. Plaques were stained with crystal violet.Virus diluent was used as a control for anti-DIII antibodies. Resultsshown in FIG. 25 demonstrate that murine polyclonal antibodies againstDIII protein are capable of neutralizing the West Nile virus

Together, these data define that domain III of West Nile virus E proteinis responsible for binding to the surface of the cells.

The disclosures of each and every publication and reference cited hereinare incorporated herein by reference in their entirety.

The present disclosure has been explained with reference to specificembodiments. Other embodiments will be apparent to those of ordinaryskill in the art in view of the foregoing description. The scope ofprotection of the present disclosure is defined by the appended claims.

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1. Pharmaceutical composition for the treatment of a flavivirusinfection in a vertebrate, the flavivirus exhibiting a flavivirusenvelope protein, the flavivirus envelope protein comprising a domainIII of the flavivirus envelope protein, the pharmaceutical compositioncomprising: a pharmaceutically effective amount of an agent thatfunctionally interferes with binding of the domain III of the flavivirusenvelope protein to a flavivirus receptor protein; and apharmaceutically acceptable carrier, vehicle or auxiliary agent, whereinthe agent comprises a polypeptide having an amino acid sequence thatexhibits at least 80% sequence identity to amino acids 350 to 390 of aflavivirus envelope sequence as set forth in SEQ ID NO: 21, and whereinthe flavivirus receptor protein is one of an integrin and a neurotensinreceptor.
 2. A vaccine for a flavivirus, the flavivirus exhibiting aflavivirus envelope protein, the flavivirus envelope protein comprisinga domain III of the flavivirus envelope protein, the vaccine comprising,as an active agent, a polypeptide having an amino acid sequence thatexhibits at least 80% sequence identity to amino acids 350 to 390 of aflavivirus envelope sequence as set forth in SEQ ID NO: 21.